RESEARCH ARTICLE Differential and directional estrogenic signaling pathways induced by enterolignans and their precursors Yun Zhu1,2, Kayoko Kawaguchi1, Ryoiti Kiyama1* 1 Advanced Biomeasurements Research Group, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki, Japan, 2 Scinet Company, 4-21-12 Takanawa, Minato-ku, Tokyo, Japan a1111111111 * [email protected] a1111111111 a1111111111 a1111111111 a1111111111 Abstract Mammalian lignans or enterolignans are metabolites of plant lignans, an important category of phytochemicals. Although they are known to be associated with estrogenic activity, cell signaling pathways leading to specific cell functions, and especially the differences among OPEN ACCESS lignans, have not been explored. We examined the estrogenic activity of enterolignans and Citation: Zhu Y, Kawaguchi K, Kiyama R (2017) their precursor plant lignans and cell signaling pathways for some cell functions, cell cycle Differential and directional estrogenic signaling and chemokine secretion. We used DNA microarray-based gene expression profiling in pathways induced by enterolignans and their human breast cancer MCF-7 cells to examine the similarities, as well as the differences, precursors. PLoS ONE 12(2): e0171390. doi:10.1371/journal.pone.0171390 among enterolignans, enterolactone and enterodiol, and their precursors, matairesinol, pinoresinol and sesamin. The profiles showed moderate to high levels of correlation (R val- Editor: Aamir Ahmad, University of South Alabama Mitchell Cancer Institute, UNITED STATES ues: 0.44 to 0.81) with that of estrogen (17β-estradiol or E2). Significant correlations were observed among lignans (R values: 0.77 to 0.97), and the correlations were higher for cell Received: October 21, 2016 functions related to enzymes, signaling, proliferation and transport. All the enterolignans/ Accepted: January 19, 2017 precursors examined showed activation of the Erk1/2 and PI3K/Akt pathways, indicating the Published: February 2, 2017 involvement of rapid signaling through the non-genomic estrogen signaling pathway. How- Copyright: © 2017 Zhu et al. This is an open access ever, when their effects on specific cell functions, cell cycle progression and chemokine article distributed under the terms of the Creative (MCP-1) secretion were examined, positive effects were observed only for enterolactone, Commons Attribution License, which permits suggesting that signals are given in certain directions at a position closer to cell functions. unrestricted use, distribution, and reproduction in any medium, provided the original author and We hypothesized that, while estrogen signaling is initiated by the enterolignans/precursors source are credited. examined, their signals are differentially and directionally modulated later in the pathways, Data Availability Statement: The microarray data resulting in the differences at the cell function level. are available in the Gene Expression Omnibus database (www.ncbi.nlm.nih.gov/geo/) with Accession No. GSE86565. Funding: This research has been supported partly by a Special Coordination Fund for Promoting Science and Technology (Encouraging Introduction Development of Strategic Research Centers), a Mammalian lignans, or enterolignans, are lignans characterized by two phenylpropanoid C - Knowledge Cluster Initiative program, a grant from 6 Kyushu Sangyo University for promoting basic C3 units, and two main types of enterolignans, enterolactone (EL) and enterodiol (ED), are technologies, and a Grant-in-Aid for Basic Areas found in the urine, plasma, saliva and/or feces of mammals as metabolites of plant lignans, from the Ministry of Education, Culture, Sports, such as matairesinol (MR), secoisolariciresinol, 7Â-hydoroxymatairesinol, lariciresinol, PLOS ONE | DOI:10.1371/journal.pone.0171390 February 2, 2017 1 / 14 Estrogenic signaling pathways for lignans Science and Technology of Japan. The funders and isolariciresinol and pinoresinol (PR), and their glycosides [1]. For example, secoisolariciresinol Scinet Company provided support in the form of diglycoside and matairesinol glycoside are metabolized by intestinal bacteria to EL, through salaries for authors [YZ and KK], but did not have secoisolariciresinol/ED or MR, respectively [2,3]. Many plant lignans are phytoestrogens, a any additional role in the study design, data collection and analysis, decision to publish, or group of plant chemicals with estrogenic activity. Enterolignans, on the other hand, have been preparation of the manuscript. The specific roles of implicated as possessing weakly estrogenic and anti-estrogenic activities and to have various these authors are articulated in the `author effects on human health, such as protective effects against cancer, osteoporosis and coronary contributions' section. heart disease through their anti-tumor, anti-oxidant and anti-estrogenic properties [1, 4±6]. Competing Interests: The authors have declared Reduced risk of breast cancer by modulating estrogen signaling was implicated after the that no competing interests exist to Scinet administration of EL and ED, and plant lignans, such as arctiin, sesamin (SE), secoisolariciresi- Company and other funders relating to nol diglucoside, lariciresinol and tracheloside [7]. Among lignans, SE has been reported as a employment, consultancy, patents, products in precursor of enterolignans with protective effects on hormone-related diseases [8]. development, or marketed products, etc., and that Estrogen is a sex hormone that plays important roles in various physiological and cellular they do not alter our adherence to PLOS ONE policies on sharing data and materials. effects and diseases through estrogen signaling [9]. A comprehensive search of estrogenic chem- icals indicates a number of chemicals with structural, functional and original variations, and the pathways involving estrogen signaling vary depending on the types of chemicals, sources/char- acteristics of cells and conditions of stimulation/signaling [10]. Among the assays for estrogenic activity, gene-expression profiling by means of DNA microarray assay is based on monitoring the estrogenic effects at the transcription level using estrogen-responsive genes with estrogen- receptor (ER) positive cells, and has been applied to a variety of chemicals [11]. A combination of DNA microarray assay with a protein assay, such as Western blotting, for monitoring specific signal mediators enabled us to understand complicated signaling pathways. Especially, the involvement of specific signaling pathways in estrogen signaling would be useful to develop anti-cancer agents and other diagnostic/therapeutic substances [11]. A number of plant lignans modulate estrogen signaling. For example, plant lignans (includ- ing their glycosides and derivatives), such as arctigenin/arctiin, guaiacin, trans-hinokiresinol, hydroxymatairesinol, 1-hydroxypinoresinol, isoguaiacin, manglieside E, MR, nordihydro- guaiaretic acid, nortrachelogenin (wikstromol), nyasol (cis-hinokiresinol), oleiferin C, oleiferin D, schizandrin, secoisolariciresinol, silibinin (silybin B) and silymarin (a mixture of flavono- lignans), show weak/moderate estrogenic/agonistic activity, while the same or other lignans, such as arctigenin/arctiin, epi-aschantin, deoxypodophyllotoxin, eleutheroside E, isolariciresi- nol, khainaoside, epi-magnolin, MR, nectandrin B, nortrachelogenin, PR, princepin, savinin, schizandrin B, SE, silibinin, syringaresinol, tracheloside, epi-yangambin and yatein, show weak/moderate anti-estrogenic/antagonistic activity (summarized in Kiyama, 2016 [12]). On the other hand, enterolignans, such as dihydroenterolactone, ED and EL, are known to show estrogenic activity [13±17]. Because of their estrogenic/anti-estrogenic activity, these lignans are often utilized for food, supplements, diagnostics and medicines. However, how to predict the estrogenic/anti-estrogenic effect of lignans is not well understood. To further explore the applications of estrogenic lignans, it is essential to understand the mechanism of action, espe- cially at the cell signaling level [12]. We examined here the estrogenic activity of enterolignans and their precursors along with the signaling pathways involved in this action. Materials and Methods Antibodies and reagents Rabbit antibodies against human cyclins D1 (#2978) and E (#4132), and cyclin-dependent kinase 4 (CDK4) (#12790), were obtained from Cell Signaling Technology (Danvers, MA). A mouse monoclonal antibody against human β-actin (#ab6276) was obtained from Abcam (Cambridge, United Kingdom). A horseradish peroxidase (HRP)-linked goat antibody against PLOS ONE | DOI:10.1371/journal.pone.0171390 February 2, 2017 2 / 14 Estrogenic signaling pathways for lignans rabbit IgG (#7074) and a horse antibody against mouse IgG (#7076) were obtained from Cell Signaling Technology. 17β-estradiol (E2; #E1132), SE (#S9314), ED (#45198), EL (#45199), MR (#40043), and PR (#40674) were obtained from Sigma-Aldrich (St. Louis, MO). ICI 182,780 was obtained from Tocris Bioscience (Bristol, United Kingdom). LY294002 was obtained from EMD Millipore (Billerica, MA). Sulforhodamine B (SRB) assay Human breast cancer MCF-7 cells were cultured in a phenol red-free RPMI 1640 medium (Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) at 37ÊC in a humidified atmosphere containing 5% CO2. The SRB assay was performed to examine cell proliferation as described previously [18]. Briefly,